Abstract

Cancer stem cells (CSC) are considered major contributor to tumor progression. Furthermore, it has been reported that standard chemotherapy regimens, as the cisplatin (CIS)-based schemes, might induce CSC phenotype in epithelial ovarian cancer (EOC) cells. Nonetheless, the influences of CSC on tumor microenvironment, as well as its role on cancer immunosurveillance remain inconclusive. Herein, we present data derived from the exposure of the CIS-sensitive EOC cell line A2780 to CIS at IC50 750 nM and half IC50 375 nM, for 5 days; after which the expression of CD44, CD24, OCT-4, citokeratin-18 (CK-18), vimentin, epithelial specific antigen (ESA), nestin and nanog was evaluated by qPCR, applying the ΔΔCt method. In parallel, protein expression of nestin and nanog was assessed by Western blot. Data were analyzed by one way ANOVA and Newmann-Keuls post-test, always using beta actin as the reference molecule. CSC markers transcripts OCT-4 (P<0.01), vimentin (P<0.001), nestin (P<0.05) and nanog (P<0.05) were upregulated by CIS 750 nM and 375 nM. At the protein level, nestin (P<0.05), but not nanog, was overexpressed only in cells treated with CIS 375 nM. The epithelial markers ESA (P<0.05) and CK-18 (P<0.001) were also overexpressed in cells exposed to both CIS concentrations. Regarding the CD44 and CD24 status, only the latter was significantly increased by CIS. We were, then, motivated to investigate the possible correlation between the CIS induced CSC phenotype and the production of immune molecules by A2780 subsequent to CIS treatment. Considering that the modulation of gene and protein expression of the molecules of interest was observed following the exposure of A2780 to CIS 375 nM, we pursued with the evaluation of cytokines production by ELISA under this experimental condition. We collected the supernatant of the CIS-treated A2780, herein designated conditioned media (CM) on days zero, three, and five, and investigated the production of TGF-β, IFN-γ, IL-17, and CXCL-1 in A2780 CM. Cytokines' concentrations were normalized by protein concentration. We found that A2780 exposure to CIS 375 nM decreased the concentration of IFN-γ (P=0.0002), IL-17 (P<0.0001), and CXCL-1 (P=0.0012), whereas TGF-β (P=0.0114) levels increased, always in a time-dependent manner. Altogether, our results suggest that the exposure of the CIS-sensitive EOC cell line A2780 to the drug might induce the CSC phenotype, and these newly developed cancer clones might be responsible for modulating tumor immune evasion. Whereas the expression of TGF-β by CSC cells has already been described, the conversion of an IL-17high/ IFN-γhigh/ TGF-βlow profile to an IL-17low/ IFN-γlow/ TGF-βhigh has not been associated to CSC phenotype yet; therefore, we postulate that it might lead, at least partially, to the switch from an antitomumoral microenvironment into a protumoral one. Chemeotatic and polarizing effects of CM on immune cells, as well as further characterization of CM are currently under investigation. To our knowledge, this is the first report correlating the effect of EOC cells exposure to cisplatin, in lower doses than those necessary to induce chemoresistance, to, apart from stimulating the acquisition of CSC phenotype by these cells, affect tumor microenvironment, and possibly compromising cancer immunosurveillance.